Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4727—Mucins, e.g. human intestinal mucin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation

Definitions

• fusion polypeptides are produced by introducing to a cell (e.g., tiansfection or transformation) a nucleic acid encoding a mucin polypeptide operably linked to a nucleic acid encoding at least a portion of an immunoglobulin polypeptide; a nucleic acid encoding an ⁇ l,3 galactosyltransferase polypeptide; and a nucleic acid encoding a ⁇ 1,6,-N-acetylglucosaminyltransferase polypeptide.
• the cell is cultured under conditions that permit production of the fusion polypeptide and the fusion polypeptide is isolated from the culture.
• Fusion polypeptides are isolated by methods known in the art. For example, the fusion polypeptides are isolated using Protein A or Protein G chromatography.
• Mass spectrometry confirmed that, the increased ⁇ -Gal epitope density was due to core 2 branching and lactosamine extensions on O-glycans of PSGL- l/mIgG 2b made in CHO cells engineered to express both C2 GnTI and ⁇ l,3GalT (Fig. 7, 8 and Table H).
• the binding of an antibody to a carbohydrate epitope is dependent on the conformation of the presented saccharide. Histo-blood group antigens have been shown to adopt different conformations in solution (hnberty, A. et al, 1995) and anti-blood group antibodies to recognize different areas of the carbohydrate determinant, the so called micro-epitopes, that were conformation-dependent (Imberty, A.
• the ⁇ Gal fusion protein is linked to a particle made of, e.g., metal compounds, silica, latex, polymeric material; a microtiter plate; nitrocellulose, or nylon or a combination thereof.
• the ⁇ Gal fusion proteins linked to a solid support are used as an absorber to remove anti- ⁇ Gal antibodies from a biological sample, such as blood or plasma.
• host cell and "recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
• an ⁇ Gal fusion polypeptides according to the invention can be chemically synthesized using methods known in the art. Chemical synthesis of polypeptides is described in, e.g., A variety of protein synthesis methods are common in the art, including synthesis using a peptide synthesizer. See, e.g., Peptide Chemistry, A Practical Textbook, Bodasnsky, Ed. Springer- Verlag, 1988; Merrifield, Science 232: 241-247 (1986); Barany, et al, Intl. J. Peptide Protein Res. 30: 705-739 (1987); Kent, Ann. Rev. Biochem. 57:957-989 (1988), and Kaiser, et al, Science 243 : 187-198 (1989).
• HAR hyperactute rejection
• transplants include but are not limited to kidney, liver, skin, pancreas, cornea, or heart.
• HAR is meant to include any antibody mediated graft rejection by the recipient.
• Hyperacute rejection of a transplanted organ occurs within seconds or minutes of exposing the organ to the recipient's circulation. The organ quickly becomes pale, and will undergo necrosis if left in the recipient's body. This hyperacute reaction occurs because of preformed antibodies against the donor organ.
• HAR is most common in multiparous women, as they have been exposed in utero to numerous non-self antigens. Recipients who have received multiple blood transfusions are also at risk.
• the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
• suppositories e.g., with conventional suppository bases such as cocoa butter and other glycerides
• retention enemas for rectal delivery.
• the concentration of PSGLl/mIgG 2 b in the supematants ranged from 150 to 200 ng/ ⁇ l, and in this particular experiment it was approximately 160 ng/ ⁇ l (FIG. 2 A, the non-absorbed column).
• the concentration of PSGLl/mIgG 2b remaining in 2, 4 and 8 ml of supernatant following abso ⁇ tion on 50 ⁇ l packed anti-mouse IgG agarose beads was 32, 89 and 117 ng/ ⁇ l, respectively.
• a hygromycin b concentration of 550, 50, and 100 ⁇ g/ml was used for CHO-K1, COS7m6 and 293T cells, respectively, and a G418 concentration of 900 ⁇ g/ml was used for CHO-K1 cells.
• the selection medium was changed every third day.
• the drug resistant clones formed after approximately two weeks. Clones were identified under the microscope and hand-picked using a pipetman. Selected colonies were cultured in 96-well plates in the presence of selection drugs for another two weeks.
• PSGL-l/mIgG 2b was produced as a dimer as indicated by the reduction to half the size upon reduction (compare Fig 7 A and B).
• the presence of ⁇ -Gal epitopes on the fusion protein made in the different cell types was detected using the GSA I IB 4 lectin (Fig. 7 B).
• a glycosylated band with a MW of 50-60 kDa, not stained by the anti PSGL-1 antibody, can be seen in the affinity purified fraction (Fig 11 A lane 4).
• This protein is probably also derived from the cell culture medium, and is adsorbed on the affinity column together with the fusion protein. This protein was removed by gel filtration, during which it eluted later (Fig. 11 A and B, lanes 7-9) than the fusion protein (Fig 11 A, B and C, lanes 5-6). Additional non-glycosylated proteins of around 50-70 kDa were removed by gel filtration (Fig. 11 B, compare lane 4 with lanes 7-9).
• the gel filtration fraction containing the highest amount of fusion protein was chosen for oligosaccharide release. As shown for clone 10 (Fig 11 A and B, lane 5), this fraction did not contain any significant amounts of contaminating proteins, glycosylated or non-glycosylated.

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• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Organic Chemistry (AREA)
• Medicinal Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Immunology (AREA)
• Biophysics (AREA)
• Molecular Biology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Genetics & Genomics (AREA)
• Engineering & Computer Science (AREA)
• Biochemistry (AREA)
• Gastroenterology & Hepatology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Zoology (AREA)
• Toxicology (AREA)
• General Chemical & Material Sciences (AREA)
• Public Health (AREA)
• Animal Behavior & Ethology (AREA)
• Transplantation (AREA)
• Pharmacology & Pharmacy (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Veterinary Medicine (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Peptides Or Proteins (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Enzymes And Modification Thereof (AREA)

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• 2003
  • 2003-08-11 US US10/638,820 patent/US7638323B2/en not_active Expired - Fee Related
  • 2003-08-11 CA CA2495743A patent/CA2495743C/en not_active Expired - Fee Related
  • 2003-08-11 AU AU2003263411A patent/AU2003263411B8/en not_active Ceased
  • 2003-08-11 EP EP03784433A patent/EP1534748A2/en not_active Withdrawn
  • 2003-08-11 NZ NZ538629A patent/NZ538629A/en not_active IP Right Cessation
  • 2003-08-11 JP JP2004527246A patent/JP4648001B2/ja not_active Expired - Fee Related
  • 2003-08-11 WO PCT/IB2003/003881 patent/WO2004015057A2/en not_active Ceased
• 2010
  • 2010-05-17 JP JP2010113707A patent/JP2010235614A/ja not_active Withdrawn

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